Phycologia Volume 57 (1), Published 6 November 2017 NI-NI-WIN 1 *, TAKEAKI HANYUDA 2,AKI KATO 3 AND HIROSHI KAWAI 2. Japan

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1 Phycologia Volume 57 (1), Published 6 November 2017 Two new species of Padina (Dictyotales, Phaeophyceae) from southern Japan, P. ogasawaraensis sp. nov. and P. reniformis sp. nov., based on morphology and molecular markers NI-NI-WIN 1 *, TAKEAKI HANYUDA 2,AKI KATO 3 AND HIROSHI KAWAI 2 1 Kyushu University Amakusa Marine Biological Laboratory, Tomioka 2231, Amakusa-Reihoku , Japan 2 Kobe University Research Center for Inland Seas, Rokkodai, Kobe , Japan 3 Takehara Fisheries Research Station, Setouchi Field Science Center, Hiroshima University, Takehara, Hiroshima , Japan ABSTRACT: Two new bistratose species of the brown algal genus Padina, P. ogasawaraensis sp. nov. and P. reniformis, sp. nov., were discovered from Ogasawara and Okinawa Islands, and Kagoshima, Japan, respectively, and characterised based on a combination of morphological and molecular analyses. Padina ogasawaraensis is characterised by a semicircular or circular yellowish-brown thallus with light to heavy calcification except at the hairlines and presence of hairlines on both surfaces in an alternating sequence. These can be seen as a broad-depressed line with a remnant of a thin red hairline at its center on the inferior surface and as a narrow or sometimes rudimentary line on the superior surface. Indusiate reproductive sori are located distally adjacent to the hairlines on the inferior surface, partially to deeply embedded in the epidermis layer. Padina reniformis is characterised by a semicircular or kidney-shaped greenish brown thallus with light to moderate calcification on both surfaces of the thallus. Hairlines are present only on one (inferior) surface and are narrow, not depressed. Indusiate tetrasporangial sori are arranged in two to three rows between the hairlines on the inferior surface and situated on the thallus surface. Molecular phylogenetic analyses using rbcl and cox3 DNA sequences placed them in distantly related clades. Padina ogasawaraensis showed a sister relationship to P. calcarea; whereas, P. reniformis was sister to P. fasciata. KEY WORDS: cox3, Molecular phylogeny, Morphology, Padina ogasawaraensis, Padina reniformis, rbcl, Taxonomy INTRODUCTION The brown algal genus Padina Adanson is widely distributed from warm-temperate to tropical seas where species occur from the lower intertidal to deep subtidal zones. The thallus is generally fan shaped with in-rolled marginal meristems, where growth is initiated. It is calcified to varying degrees at least on the superior surface (facing the in-rolled margin). Some species, however, show an uncalcified Vaughaniella stage (creeping rhizomes, also known as the Dictyerpa stage) with a single apical cell from which the fan-shaped thalli develop (Børgesen 1950; Cribb 1951; De Clerck & Coppejans 1997). Fronds grow either upright or decumbent and are composed of two to many (up to 20) cell layers, depending on the species. Phaeophycean hairs in concentric rows called hairlines may occur on one or both thallus surfaces, except for P. glabra Gaillard, whose thalli are without hairlines (Gaillard 1966; Wynne & De Clerck 1999). Their location and disposition are generally related to the position and arrangement of the rows of reproductive sori, both being important for species identification. The life cycle is an alternation of diplohaplontic isomorphic generations. Padina is the second most species-rich genus in the order Dictyotales after Dictyota and includes 52 currently recognised species (Guiry & Guiry 2017). Of these, 17 species were reported in Japan (Ni-Ni-Win et al. 2008, 2010, 2011a, 2012; * Corresponding author (nini.niniwin@gmail.com). DOI: / Ó 2018 International Phycological Society Yoshida et al. 2000). In the past, species of Padina were taxonomically challenging due to morphological plasticity coupled with poor understanding of reliable morphological characters. However, the advent of molecular markers and phylogenetic tools helped us to assess and recognise several reliable morphological characters for species delineation and morphological plasticity (e.g. thallus shape, size, colour, cell size and structure, thallus thickness, etc.) caused by environmental conditions and age of the individual (Ni-Ni- Win et al. 2011a). All previous studies on Padina in Japan and globally were exclusively based on morphology until recent studies by Ni-Ni-Win et al. (2008, 2010, 2011a, b, 2012, 2013) that used a combination of detailed morphological and multigene molecular data. Those studies have clarified the systematics of the genus as well as our understanding of the boundaries of reliable morphological characters for species delineation. In addition, many new species and new records were reported from Japan as well as from the Indo-Pacific regions and the Mediterranean Sea. While Padina species diversity in Japan more than doubled, unexpected species diversity was also observed. Only eight Padina species were reported in Japan before Recently, other comprehensive molecular studies of Padina have further confirmed the existence of cryptic species, revealing remarkable species diversity both globally and regionally (Silberfeld et al. 2013; Diaz-Martinez et al. 2016). In the present study, two new Padina species are reported from southern Japan based on molecular phylogenetic analyses using chloroplast-encoded rbcl and mitochondrial cox3 20

2 Ni-Ni-Win et al.: Padina ogasawaraensis sp. nov. and P. reniformis sp. nov. 21 gene sequences in combination with morphological observations, and their descriptions are presented. MATERIAL AND METHODS Padina specimens used in this study were newly collected in a wide range of localities on Chichijima, (Ogasawara Is., Tokyo), Okinawa Is. (Okinawa) and Ishikaki (Minamikyushu, Kargoshima), Japan (supplementary data Table S1). Selected voucher specimens used for morphological and molecular studies were deposited in the herbaria of Kobe University Research Center for Inland Seas and the Graduate School of Science, Hokkaido University (SAP). Type specimens of P. australis Hauck (L ), P. distromatica Hauck (L ), P. dubia Hauck (L ), P. somalensis Hauck (L ), P. tetrastromatica Hauck (L ) (Hauck No. 68), P. haitiensis Thivy (MICH, Taylor 20987), P. perindusiata Thivy (MICH, Taylor 1356) and P. japonica Yamada (SAP 9268), borrowed from Naturalis, Leiden, The Netherlands (L), the Herbarium of University of Michigan (MICH), and the Herbarium of Graduate School of Science, Hokkaido University (SAP), were also examined. Herbarium abbreviations follow Thiers (2011). Specimens were hand sectioned for anatomical observations and the sections mounted on glass slides in Karo syrup/seawater. Photomicrographs were taken using a VB-7010 Digital Camera (Keyence, Tokyo, Japan) attached to a BX-51 microscope (Olympus, Tokyo, Japan). DNA extraction, polymerase chain reaction (PCR) amplifications and sequencing were carried out as described in Ni-Ni-Win et al. (2008, 2010). Three new rbcl and cox3 sequences generated in this study were deposited at DDBJ and listed in Table S1, together with additional previously published sequences (Ni-Ni-Win et al. 2008, 2010, 2011a, b, 2012, 2013). Twenty rbcl and 18 cox3 sequences of Padina species generated by Silberfeld et al. (2013) were downloaded from GenBank and analysed with our sequences. These represented either already reported species or unidentified species. Six taxa Dictyota dichotoma (Hudson) J.V.Lamouroux AB358934, Dictyopteris muelleri (Sonder) Reinbold JQ364137, Distromium didymothrix Allender & Kraft EU579948, Homoestrichus sp. EU579951, Lobophora sp. AB and Stypopodium sp. AB (Dictyotales) were used as out-groups in rbcl analyses (Table S1). The same out-group taxa, except for D. didymothrix and Homoestrichus sp., together with Zonaria sp. AB665391, were used for cox3 analyses since no cox3 sequence was available for those two taxa. Dictyopteris muelleri, D. dichotoma and Stypopodium sp. were used for the concatenated analysis since sequences of both genes (rbcl and cox3) were available only for these three species. Sequences were aligned with Clustal X (Thompson et al. 1997) and then manually adjusted by eye. To check the positions/clusters of the specimens attributed to a species, as well as for checking the congruence of tree topologies, three alignments (i.e. rbcl, cox3 and rbclþcox3 combined) were created to construct phylogenetic trees. Phylogenetic trees were obtained using maximum likelihood (ML) and Bayesian inference (BI) analyses, as implemented, respectively, in RAxML-HPC2 (Stamatakis 2014) and MrBayes v3.2.2 (Ronquist & Huelsenbeck 2003) on the CIPRES Science Gateway v3.3 (Miller et al. 2010). ML analyses were carried out using the GTRþG model and 1000 rapid bootstrap replicates, with other parameters being the default settings for both bootstrapping and best-tree searching phases. BI analyses were initiated with a random starting tree, and two parallel simultaneous Markov chain Monte Carlo runs were performed. Each run consisted of four chains for 10,000,000 generations with sampling one tree every 1000 generations. The first 10,000 trees sampled were discarded as burn-in, based on the stationarity of -ln L as assessed using Tracer v1.6 (Rambaut et al. 2014). A consensus topology and posterior probability values were calculated with the remaining trees. The resulting phylogenetic trees were visualised with FigTree v1.3.1 (Rambaut 2009), and Adobe Illustrator CS2 was used for figure editing. RESULTS Molecular analyses The rbcl alignment consisted of 1319 characters of 125 sequences representing 39 Padina species and six out-group taxa, including three new sequences of Japanese specimens. The cox3 alignment consisted of 748 characters of 113 sequences representing 37 Padina species and five out-group taxa, including three new sequences of Japanese specimens (Table S1). A representative sequence of each species and three out-group taxa was used for the combined analyses. Some species were represented by multiple specimens, resulting in a 2067-base-pair (bp) combined alignment of 46 sequences representing 39 Padina species in total. All alignments were unambiguous without gaps. ML and BI analyses using these three alignments showed almost identical tree topologies, except for the position of some clades that received no or low supports. The ML tree inferred from the concatenated data set is shown in Fig. 1, and those from the separate data set of rbcl and cox3 are shown in supplementary data Figs S1 and S2, respectively, with ML bootstrap values (. 50%) and BI posterior probabilities (. 0.70) plotted near the root of each clade. In the molecular analyses, the newly collected Japanese specimens showed different sequences that formed two distinct and well-supported lineages in both separate and combined analyses (Figs 1, S1, S2). They were morphologically different from each other as well as from already described Padina species. They were assigned below as two new species, P. reniformis sp. nov. and P. ogasawaraensis sp. nov., respectively. A total of four specimens of P. reniformis showed identical sequence in both rbcl and cox3, and thus a single sequence was used as a representative. Likewise, seven specimens of P. ogasawaraensis produced identical sequences in rbcl, but they were 0 1 bp different in cox3. A specimen from New Caledonia, assigned as P. sp. 3 in Silberfeld et al. (2013), grouped with Japanese P. ogasawaraensis with high supports in all analyses. Sequence divergences between them was very low, 0.7% in rbcl and 0.1% 1.9% in cox3. The New Caledonian specimen was considered conspecific with P. ogasawaraensis. Likewise, three sequences described as P. sp.

3 22 Phycologia, Vol. 57 (1) Fig. 1. Maximum likelihood phylogeny of Padina inferred from the concatenated data set of rbcl and cox3 sequences. Numbers near nodes indicate ML bootstrap values (. 50%) and Bayesian posterior probabilities (. 0.70). Asterisks indicate full support values (100/1.00). 1, P. sp. 2 and P. sp. 5 in Silberfeld et al. (2013) clustered in the clades of P. moffittiana Abbott & Huisman, P. calcarea Ni-Ni- Win, S.G.A.Draisma, W.F.Prud homme van Reine & H.Kawai and P. boryana Thivy, respectively, with high and full support values. Genetic divergences within each species clade was 0% 0.6% in rbcl and 0.2% 4.6% in cox3, 0% 1.4% in rbcl and 0.6% 6.6% in cox3and0% 0.4% in rbcl and 0.3% 2.1% in cox3, respectively. Sequence divergences within each of these clades fell within the range of intraspecific sequence divergences of Padina species (see Ni-Ni-Win et al. 2008, 2010, 2011a, b, 2013). An exception was P. calcarea, where intraspecific divergence was slightly greater than the interspecific sequence divergence of some sister species (e.g. divergence between P. sanctae-crucis Børgesen and P. japonica is 0.83% 1.06% in rbcl and 4.5% in cox3 and between P. ryukyuana Y.P.Lee & Kamura and P. sulcata Ni-Ni-Win, S.G.A.Draisma & H.Kawai 1.1% 1.36% in rbcl and 3.49% 3.90% in cox3). Padina sp. 1, P. sp.2andp. sp.5werehereconsidered conspecific with the identified species in their respective clades until their detailed morphological observations/comparisons are available to confirm their identities. All phylogenetic analyses revealed strongly supported sister-species relationships of P. reniformis with P. fasciata Ni-Ni-Win, M.Uchimura & H.Kawai and P. ogasawaraensis with P. calcarea.

4 Ni-Ni-Win et al.: Padina ogasawaraensis sp. nov. and P. reniformis sp. nov. 23 Taxonomic description Padina ogasawaraensis Ni-Ni-Win, T.Hanyuda, M.Uchimura & H.Kawai sp. nov. Figs 2 18 DIAGNOSIS: Thalli semicircular or circular with slight to heavy calcification and attached by a stupose base with a short stipe. Thalli composed of two cell layers throughout. Concentric hairlines in alternate sequence between both surfaces of the thallus, forming a broad, depressed, uncalcified line with a red narrow hairline at its center on the inferior surface and a narrow, not depressed inconspicuous line on the superior surface. Plant dioecious; both oogonial and tetrasporangial sori forming continuous or broken lines, located distally adjacent to the hairlines on inferior surface, partially embedded in the epidermis layer, occurred with a persistent indusium; both oogonia and tetrasporangia obovate. Antheridial sori forming discontinuous lines or patches located just above the hairlines on the inferior surface, without indusium. HOLOTYPE: SAP (collected by H. Kawaii, 11 May 2007), Fig. 3, and deposited at the Herbarium of the Graduate School of Science, Hokkaido University (SAP). TYPE LOCALITY: Chichijima, Ogasawara Is., Tokyo Prefecture, Japan. ETYMOLOGY: Species epithet refers to one of the collection sites where the species was found. HABITAT: Subtidal zone to 15-m depth. OTHER SPECIMENS EXAMINED: See supplementary data. MORPHOLOGY: Thalli were semicircular or circular, wider than tall, up to 12 cm wide and 8 cm tall, with entire margin, rarely split and solitary when young but becoming shallowly to deeply split into fanshaped lobes when old, yellowish or pale to dark brown, and attached by a stupose base with a short stipe (Figs 2 5). Long fibrous hairs occurred from the base to 1 2 cm upward (Figs 2 5). Calcification was slight to moderate on the inferior surface of the thallus except at the hairlines but moderate to heavy on the superior thallus surface, forming yellowish or brownish white colour (Figs 2 7). The thallus was composed of two cell layers throughout the entire body (Fig. 8), measuring lm at the basal portion and lm thick elsewhere. Cells of the superior surface were times taller than those of the inferior surface (Fig. 8). Concentric hairlines on the inferior thallus surface were broad (0.7 1 mm wide), slightly depressed and uncalcified (Figs 7, 9, 11 14) sometimes with a red, narrow hairline at its center in some portions of the hairlines (Fig. 9), forming uncalcified stripes on the finely continuous calcified thallus surface. The hairlines on the superior surface were narrow and not depressed (Fig. 10) or sometimes rudimentary or inconspicuous. Hairlines on the two surfaces were arranged in alternate sequence between both thallus surfaces almost at equal distance when the hairlines on both surfaces are viewed together, measuring 3 5 mm apart on each surface (Fig. 15). The species was dioecious, and reproductive sori were found only on the inferior surface of the thallus. Tetrasporangial sori formed mostly continuous lines (Figs 11 13); whereas. oogonial sori formed continuous or broken lines (Fig. 14). Both were located above the hairlines of the inferior surface (Figs 11 15) (i.e. distally adjacent to the hairlines), but additional sori as small patches or broken lines were sometimes found below the hairlines or beside the normal sori (Fig. 12). Antheridial sori formed discontinuous lines or patches situated just above the hairlines of the inferior surface and had no indusia (Fig. 16). Tetrasporangial sori were slightly broader than oogonial sori (Figs 11 13, 18). Both sori were partially embedded in the epidermis layer, but sometimes oogonial sori formed on the thallus surface (not embedded in the cuticle layer) (Fig. 14) and were covered with a persistent indusium (Figs 13, 17, 18). After spore release, they left empty spaces as an empty continuous line parallel to the hairlines. Both appeared as two uncalcified brown lines (Fig. 12). Oogonia and tetrasporangia were both obovate (Figs 17, 18), lm wide and lmlongand lm wide and lm long, respectively. REMARKS: The species showed two types of thallus appearance. One type had slightly broader and conspicuous hairlines on the inferior surface, a wider space between them (c mm apart) (Figs 4, 5) and broader sporangial sori (Figs 11 13, 18). The other type showed a more yellowish brown colour and had slightly narrower hairlines on the inferior surface, a narrower space between them (ca. 2 3 mm apart) (Figs 2, 3) and narrower sori that mostly formed discontinuous lines (Fig. 14). The former type was assumed to be tetrasporophytic since those characters only occurred in tetrasporophytes, and the latter type represented gametophytes. Overall morphological features of the species were similar to the recently described species P. undulata Ni-Ni-Win, S.Arai & H.Kawai. There were only two distinct morphological characters between the two species: (1) the structure of hairlines on the inferior surface [a broad, slightly depressed, uncalcified line with a red, narrow hairline at its center in P. ogasawaraensis (Fig. 9) vs a broad, depressed, uncalcified line with a red, narrow hairline at its upper end in P. undulata]; and (2) the oogonial and tetrasporangial sori disposition (partially embedded in the epidermis layer in P. ogasawaraensis vs on the thallus surface or sometimes slightly embedded in the epidermis layer in P. undulata). Padina reniformis Ni-Ni-Win & H.Kawai sp. nov. Figs DIAGNOSIS: Thalli reniform, up to 7 cm wide and 5 cm tall, with entire margin, solitary, not split into small lobes, with slight to heavy calcification on thallus surfaces, attached by a stupose base with a short stipe; Vaughaniella stage present. Thalli composed of two cell layers throughout. Concentric hairlines narrow, not depressed, sometimes inconspicuous, forming only on the inferior surface. Tetrasporangial sori formed continuous lines arranged in two to three rows between the hairlines; covered with a persistent indusium. Gametophyte not observed. HOLOTYPE: SAP (collected by Ni-Ni-Win, 25 May 2005), Figs 19, 21 24, and deposited at the Herbarium of Graduate School of Science, Hokkaido University (SAP). TYPE LOCALITY: Ishikaki, Minamikyushu city, Kagoshima Prefecture, Japan. ETYMOLOGY: Species epithet refers to the kidney-shaped thallus structure. HABITAT: Lower intertidal zone to subtidal zone of 4 6-m depth. OTHER SPECIMENS EXAMINED: Japan: Kagoshima Pref., Minamikyushu city, Ishikaki (leg. Ni-Ni-Win & A. Kato, 25 May 2005, NNW79 83; all specimens were deposited at the Herbarium of Kobe University Research Center for Inland Seas). MORPHOLOGY: Thalli were kidney-shaped and wider than tall (up to cm), with an entire margin. They were solitary, not split into small segments and attached by a short stipe with a stupose base (Fig. 19). Calcification was slight or negligible on the inferior thallus surface (Figs 19, 21, 22, 24); whereas, it was moderate to heavy on the superior surface. The inferior surface of the thallus was yellowish green to greenish brown, and the superior surface was white to yellowish white. A Vaughaniella stage was present (Fig. 19). The thallus was composed of two cell layers throughout and lm thick from the margin to the basal portion (Fig. 20). Concentric hairlines were rather narrow, not depressed, placed on the thallus surface and found only on the inferior surface (Figs 19, 21 23). They were sometimes inconspicuous and 3 4 mm apart (Figs 19, 21, 22). Tetrasporangial sori formed more or less continuous lines that were

5 24 Phycologia, Vol. 57 (1) Figs 2 8. External and internal morphology of Padina ogasawaraensis sp. nov. Figs 2 5. Habits of gametophytes and tetrasporophytes. Inferior surface (IF) (double arrowheads) and superior surface (SP) (arrowheads). Bar ¼ 1 cm. Figs 2, 3. Gametophytes.

6 Ni-Ni-Win et al.: Padina ogasawaraensis sp. nov. and P. reniformis sp. nov. 25 distally very close to the hairlines (Figs 19, 21 23), and one to two additional sori were also found beside the regular one; this resulted in two to three rows of sori serially arranged between two hairlines (Figs 19, 21, 24). Additional sori might occur as small patches spread irregularly among the continuous sori (Fig. 22). They were located on the thallus surface and covered with a persistent transparent indusium (Figs 21, 23, 24). After spores were released, they left empty spaces as ruptured small bags arranged in a continuous or discontinuous line on the thallus surface (Figs 21, 22). Gametophytes were not observed. REMARKS: The species was similar to P. crassa Yamada in thallus appearance and hairline structures, and to P. japonica in the formation of a Vaughaniella stage and the position and arrangement of sporangial sori, respectively. However, P. reniformis differed from P. crassa and P. japonica as well as from other related species in a combination of some features, such as kidney-shaped thalli, formation of hairlines only on the inferior surface and deposition of sporangial sori arranged in two to three rows between the hairlines, which were formed on the thallus surface (i.e. not partially embedded in the cuticle layer). DISCUSSION Several recent comprehensive studies on Padina taxonomy based on morphology and molecular analyses of chloroplastencoded rbcl and mitochondrial cox3 sequences revealed the occurrence of many new species and new records in tropical and warm temperate Pacific regions including the eastern Indian Ocean and Mediterranean Sea (Ni-Ni-Win et al. 2008, 2010, 2011a, b, 2012, 2013). Those studies indicated unexpected high species diversity in those areas. In addition, a recent comprehensive study on biodiversity, global distribution and molecular phylogeny of worldwide Padina species also indicated the existence of cryptic species in some lineages (e.g. P. antillarum/p. tetrastromatica, P. gymnospora, P. melemele/p. fraseri) distributed in tropical to warm temperate Atlantic and Indo-Pacific regions (Silberfeld et al. 2013). Likewise, Diaz-Martinez et al. (2016) reassessed Mexican Padina based on DNA-assisted taxonomy together with morphological data and revealed the existence of cryptic and polymorphic species, demonstrating a higher species diversity than previously estimated in Mexican waters. They also suggested the critical need for a thorough examination of type specimens for the correct assignment of scientific names to the genetic lineages and the establishment of new species. Our molecular analyses in combination with morphological investigations in the present study demonstrated the existence of two additional new species, P. ogasawaraensis sp. nov. and P. reniformis sp. nov., in Ogasawara and Okinawa islands and in Kagoshima, respectively. Molecular phylogenetic analyses using both separate and combined data sets of rbcl and cox3 sequences demonstrated their placement in two distantly related lineages. Padina ogasawaraensis is sister to P. calcarea, and P. reniformis is sister to P. fasciata. Bothsister-group relationships showed high bootstrap supports. Detail morphoanatomical examinations showed no morphological matches of these two species to the previously reported Padina species (Table 1). They were bistratose throughout the entire thallus. The new species result in 25 distromatic species for Padina so far. Of these, 16 species were included in the molecular analyses, and they are all distinctly placed in all molecular analyses using both rbcl and cox3. The morphology of the remaining seven species was compared with the two new species (Table 1), and their morphological comparison, together with two genetically closest species (P. calcarea and P. fasciata), was included in supplementary data Table S2. The two new species are distinguishable from all previously reported distromatic species as well as from other members of the genus by their respective distinctive characters. Padina ogasawaraensis has a unique feature of the inferior surface hairline structure: a thin red hairline located at the center of a c. 300-lm broad, slightly depressed, uncalcified line (Fig. 9). This differs from the other species of Padina, whose inferior surface hairlines either have a thin red line close to the upper end of a broad, depressed, uncalcified line or only have a thin line placed on the thallus surface (i.e. only a thin undepressed line located on the thallus surface, e.g. the hairlines of P. reniformis; Figs 19, 21 23). Padina reniformis can be distinguished by a combination of some features, such as a kidneyshaped thallus, position of thin undepressed hairlines formed only on the inferior surface of the thallus and arrangement of sporangial sori in two to three rows between the hairlines. Padina ogasawaraensis is similar to the recently described P. okinawaensis and P. undulata in thallus appearance, especially in thallus colour (yellowish brown to dark brown), thallus structure and arrangement of uncalcified brown hairlines and calcified glabrous zones in alternate sequence on the inferior surface. However, in addition to other distinct morphological characters (Table 1), they can be distinguished by the inferior surface hairline structure (thin red hairline located at the center of the broad, slightly depressed line in P. ogasawaraensis vs thin red hairline placed at the upper end of a broad, depressed line in P. okinawaensis and P. undulata). They also differ in the deposition of reproductive sori (sori deeply embedded in the epidermis layer, which form a continuous line distally parallel to the hairline in P. ogasawaraensis vs sori shallowly embedded in the epidermis layer or under the calcium flake, which form mostly broken lines in the other two species). Molecular phylogenetic analyses also revealed their differences, placing them in distantly related clades in all analyses using rbcl and cox3 sequences. A total of more than 50 specimens of P. ogasawaraensis collected from Okinawa and Ogasawara islands were morphologically thoroughly examined. They showed similar morphology, especially in the unique features of inferior hairline structure, yellowish brown thallus nature with the alternate sequence of broad, slightly depressed, uncalcified hairlines and calcified glabrous zones. However, some morphological variations were found between gametophytes and sporophytes. This is exceptional since Padina species are generally isomorphic. Hairlines on the inferior surface of the sporophytes are Figs 4, 5. Tetrasporophytes. Fig. 6. Superior surface of the thallus with continuous calcification. Bar ¼ 1 cm. Fig. 7. Inferior surface of the thallus, showing the alternate sequence of broad, slightly depressed, uncalcified hairlines (arrowheads) and calcified glabrous zones. Bar ¼ 5 mm. Fig. 8. Transverse section of the middle portion of the thallus. Bar ¼ 50 lm.

7 26 Phycologia, Vol. 57 (1) Figs External and internal morphology of Padina ogasawaraensis sp. nov. (continued) Fig. 9. Detail of an inferior surface hairline (arrowhead) in surface view with a remnant of hairs in line (arrow) at the center of a broad, depressed, uncalcified area. Bar ¼ 0.4 mm. Fig. 10. Detail of a superior surface hairline (arrow) in surface view. Bar ¼ 400 lm. Fig. 11. Surface view of the inferior surface of a tetrasporophyte, showing hairlines (arrowheads) and tetrasporangial sori (double arrowheads). Bar ¼ 5 mm. Fig. 12. Surface view of the inferior surface of a tetrasporophyte, showing the relationship of hairlines (arrowheads), additional tetrasporangial sori (double arrowheads) and empty sori (arrow). Bar ¼ 5 mm. Fig. 13. Detail of a hairline (arrowhead) and sporangial sori (double arrowhead) in surface view. Bar ¼ 1 mm.

8 Ni-Ni-Win et al.: Padina ogasawaraensis sp. nov. and P. reniformis sp. nov. 27 Figs 19, 20. External and internal morphology of Padina reniformis sp. nov. Fig. 19. Habits of three tetrasporophytes, showing a Vaughaniella stage (double arrowhead), hairlines (arrowheads), tetrasporangial sori (short arrows) and fibrous hairs (long arrow) at the base. Bar ¼ 1 cm. Fig. 20. Transverse section of the middle portion of the thallus. Bar ¼ 50 lm. broader and more depressed than those of gametophytes, and the distance between hairlines are broader than those in the gametophytes (c mm vs 2 3 mm). In addition, reproductive sori form a broader and more or less continuous line in the sporophytes compared to the gametophytes in which sori lines are narrower. However, these morphological differences were not reflected in the genetic variation (i.e. all examined specimens had identical rbcl and cox3 sequences). Nonetheless, additional specimens are necessary for further morphological examination to confirm whether these characters are characteristic for the different life-history stages. Similarly, P. reniformis resembles P. crassa in thallus structure and hairline disposition and P. japonica in the formation and structure of a Vaughaniella stage and Fig. 14. Surface view of the inferior surface of a gametophyte, showing the relationship of hairlines (arrowhead) and oogonial sori (double arrowheads). Bar ¼ 5 mm. Fig. 15. Surface view of the inferior surface of a fresh specimen (tetrasporophyte) with alternate hairlines (arrowheads on IF surface; arrow on SP surface) and sporangial sori (double arrowheads). Bar ¼ 1 mm. Fig. 16. Detail of antheridial sori (arrowhead) in section and in surface view (inlet). Bar ¼ 50 lm. Fig. 17. Detail of oogonial sori (arrow) with indusium (arrowheads) in surface view. Bar ¼ 200 lm. Fig. 18. Detail of tetrasporangial sori (arrow) with indusium (arrowheads) in surface view. Bar ¼ 200 lm.

9 28 Phycologia, Vol. 57 (1) Table 1. Comparison of morphological features among bistratose species that are not included in the molecular analyses and the two new Padina species. 1 Characters P. condominium Kraft P. distromatica P. fernandeziana Skottsberg & Levring P. haitiensis Vegetative characters Thallus Shape flabelliform flabelliform flabelliform circular Growing nature NA NA erect semiprostrate Size cm ~ 10 cm ~ 20 cm ~ 5cm Colour (IF/SP) NA NA brown-dark brown/na greyish brown/light brown Margin NA entire split entire split entire Calcification IF/SP light/light light/moderate no/no heavy/heavy surfaces Vaughaniella stage NA absent NA absent Rhizoid-like hairs on NA absent NA absent thallus Thickness of cell layers Structure Hairlines Position (surfaces; arrangement) Structure (IF/SP) IF cells thinner than SP cells NA IF cells two times thicker than SP cells IF cells 0.66 times as deep as SP cells both surfaces; alternate both surfaces; alternate both surfaces; alternate both surfaces; alternate conspicuous/conspicuous; narrow, not depressed/ narrow, not depressed conspicuous/ inconspicuous (rudimentary); broad/ narrow conspicuous/conspicuous; NA conspicuous/conspicuous; narrow, depressed/ narrow, depressed Alternate HLs equal distance NA equal distance equal distance arrangement Distance on each NA NA ~ 3 mm mm surface Reproductive characters Reproductive systems monoecious dioecious? NA dioecious? Sporangial sori Position IF IF SP IF Structure NA NA broad narrow No. of rows between one? two one to two (three) one HLs Arrangement NA; above the HLs NA; abutting HLs broken lines small groups; between HLs broken lines; in the middle of HLs Location NA NA partially embedded in the on thallus surface cuticle layer Indusium present absent present 3 present 3 Fertile zone alternate 4 successive 5 successive 5 alternate 4 References Kraft (2009) Hauck (1887); examination of type specimen Levring (1941); Gaillard (1973) Taylor (1960); examination of type specimen 1 IF, inferior surface; SP, superior surface; HLs, hairlines; NA or, not available (not described). 2 Sometimes with additional broken line. 3 Indusium present in tetrasporophyte and female gametophyte but absent in male gametophyte. 4 Fertile zone is separated by sterile zone when both surfaces are viewed together. 5 Sterile zone is absent. arrangement of hairlines and sporangial sori. They, however, are respectively distinctive in both genetic (rbcl, cox3 and their concatenated data; Figs 1, S1, S2) and some morphological features. Padina reniformis differs from P. crassa in its bistratose nature of the thallus, the presence of a Vaughaniella stage and hairline position only on one surface and the arrangement of two to three sori between the hairlines. Padina reniformis differs from P. japonica in its kidney-shaped thallus structure and the position of the hairline only on one surface. Padina jonesii was reported to have a kidney-shaped thallus (Tsuda 1972), one of the distinctive characters of P. reniformis, but the former differs from the latter in many characters, such as a prostrate thallus, position of hairlines on both surfaces, the presence of small groups of rhizoid-like hairs on the inferior surface, disposition of reproductive sori distally just above the hairlines, and so on. In Padina, concentric phaeophycean hairlines are present in all members either as a scar of hairs or as a remnant of hairs except in P. glabra, whose thalli were reported to be without hairlines (Gaillard 1966; Wynne & De Clerck 1999). The hairline location and disposition are important characters for Padina species delineation. Presence of hairlines in an alternate sequence in either equal or unequal distance between both surfaces is a common feature in all members, and there were only three species known, P. boryana, P. calcarea and P. melemele, in which hairlines are present only on one surface (inferior) until the discovery of the new species P. reniformis. These four species differ in both molecular (Figs 1, S1, S2) and morphological features. A combination of kidney-shaped thallus, hairlines posi-

10 Ni-Ni-Win et al.: Padina ogasawaraensis sp. nov. and P. reniformis sp. nov. 29 Table 1. Extended P. jonesii Tsuda P. ogasawaraensis sp. nov. P. perindusiata P. plumbea (Areschoug) Levring P. reniformis sp. nov. tioned only on one surface and formation of a Vaughaniella stage (plus disposition of sori in two to three rows between hairlines for mature specimen) make P. reniformis a unique species that can easily be identified in the field. Molecular phylogenetic analyses showed a clade of a New Caledonian specimen named P. sp. 3 in Silberfeld et al. (2013) and Japanese specimens of P. ogasawaraensis with very low sequence divergence in both rbcl (0.7%) and cox3 (1.8% 1.9%). Their low sequence divergence is considered not to exceed intraspecific genetic variation; therefore, the New Caledonian sample is tentatively placed under the name of P. ogasawaraensis until the specimen is available for detailed morphological observation and comparison with Japanese specimens. Similarly, sequences assigned as P. sp. 1 and P. sp. 2 collected, respectively, from Fowlers Bay, South Australia, and Lifou, New Caledonia (Silberfeld et al. 2013), showed identical (rbcl) and almost identical (cox3, 1 bp difference) sequences with our Australian P. moffittiana from Rottnest Island and P. calcarea from Lizard Island, respectively. Ni-Ni- reniform circular-semicircular semicircular-flabelliform flabelliform kidney- semicircular prostrate erect erect erect Erect ~ 10 cm 6 10 cm ~ 10 cm ~ 20 cm 6 8 cm light brown yellowish brown/ yellowish white NA brown/brown brownish green/pale white NA entire split entire split split entire heavy/heavy light-moderate/moderateheavy light/light no/no no or light/moderate NA present absent NA present present absent absent absent absent NA IF cells times thinner than SP cells IF cells as deep as SP cells IF cells two times thicker than SP cells IF cells slightly thinner than SP cells both surfaces; alternate both surfaces; alternate both surfaces; irregular both surfaces; irregular only on IF surface conspicuous/conspicuous; broad, depressed/na conspicuous/conspicuous; broad, depressed/ narrow, not depressed conspicuous/conspicuous; narrow, not depressed/ narrow, not depressed inconspicuous / inconspicuous; NA inconspicuous/-; narrow, not depressed/ equal distance equal distance irregular irregular? 3 4 mm 2 4 mm irregular ~ 3 mm? 3 4 mm gametophytes unknown dioecious NA NA gametophytes unknown IF IF IF SP IF narrow narrow broad NA narrow one one 2 two to three two? two to three continuous lines; above the HLs continuous lines; above the HLs continuous broken lines; between HLs broken lines patches; between HLs continuous broken lines; between HLs NA partially embedded in the on thallus surface slightly embedded in the on thallus surface cuticle layer cuticle layer absent present 3 present present present alternate 4 alternate 4 successive 5 successive 5 successive 5 Tsuda (1972) this study Taylor (1960); examination of type specimen Levring (1940); Gaillard (1973) this study Win et al. (2013) performed a detailed morphological and molecular examination of Australian Padina samples from Rottnest, Heron and Lizard islands. They found both genetic and morphological similarities of Australian specimens to P. moffittiana from Japan and Hawaii (including the type) and to P. calcarea from Indonesia and Palau (including the type). This suggests the geographical extension of P. moffittiana and P. calcarea to Australia. Although the Australian (P. sp. 1) and New Caledonian (P. sp. 2) specimens used in Silberfeld et al. (2013) were not available for morphological examination and comparison, we consider them as P. moffittiana and P. calcarea, respectively, based on their identical sequences with the Australian samples that have been genetically and morphologically examined and compared with type specimens. This also applies to the specimen designated as P. sp.5 in Silberfeld et al. (2013). A sequence of P. sp. 5 from Sri Lanka is genetically identical with those of P. boryana from Myanmar and the Philippines in rbcl, and they were only 1 bp different in cox3. Therefore, we consider P. sp. 5 to be P. boryana.

11 30 Phycologia, Vol. 57 (1) Figs Morphology of Padina reniformis sp. nov. (continued). Fig. 21. Surface view of the inferior surface of a tetrasporophyte, showing the relationship of hairlines (arrowheads) and tetrasporangial sori (arrows). Bar ¼ 500 lm. Fig. 22. Surface view of the inferior surface of a tetrasporophyte with hairlines (arrowheads) and empty tetrasporangial sori (arrows). Bar ¼ 1 mm. Fig. 23. Detail of a hairline (arrowhead) and tetrasporangial sori (short arrow) with indusium (long arrow) in surface view. Bar ¼ 100 lm. Fig. 24. Surface view of the inferior surface of tetrasporangial sori (short arrows) with indusium (long arrows). Bar ¼ 500 lm. Congruent with several previous studies of Padina based on both morphological and molecular data by Ni-Ni-Win and coauthors, Silberfeld et al. (2013) and Diaz-Martinez et al. (2016), the present study has furthermore confirmed unexpected species richness in the northwestern Pacific, especially in southern Japan. Only eight species were reported from Japan (Yoshida et al. 2000) until recent comprehensive studies on the genus Padina by Ni-Ni-Win et al. (2008, 2010, 2011a, b, 2012, 2013). Ni-Ni-Win et al. increased the number of Japanese Padina species to a total of 19 species including the two new species in the present study, showing a nearly 150% increase of local species diversity. Okinawa is the most species-rich area in Japan. All Japanese Padina species were found in Okinawa, except P. boryana, P. thivyae and P. reniformis. Seven new species and two new records were recently reported for this area. Previous underestimation of Padina species diversity was probably driven by poor understanding of morphological plasticity and reliable morphological characters for species delineation coupled with the lack of molecular data. In addition, misinterpretation of the original descriptions (e.g. interpretation of terminology for inferior/superior or upper/lower or dorsal/ventral thallus surfaces related to the in-rolled margins in Padina; see details in Ni-Ni-Win et al. 2010) of

12 Ni-Ni-Win et al.: Padina ogasawaraensis sp. nov. and P. reniformis sp. nov. 31 type specimens is another cause of erroneous identification or underestimation of biodiversity. Many Padina species may remain to be discovered. Geographically, P. ogasawaraensis was widely found in the Ogasawara and Okinawa islands but not in other places in Japan or outside Japan. However, considering a New Caledonian specimen being P. ogasawaraensis based on genetic similarity, this species probably has a wider distributional range. Padina reniformis was found only in one place in Kagoshima despite extensive sampling throughout Japan, Hawaii, the Mediterranean Sea and the eastern Indo-Pacific. This species is possibly endemic to Japan. However, additional sampling in other regions might be able to confirm either its endemism in Japan or greater distribution outside Japan. ACKNOWLEDGEMENTS We are thankful to Dr S.G.A. Draisma for his valuable comments and improving the English. 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